Allograft rejection is mediated by the recipient?s immunological response to donor antigen that is initiated and coordinated by CD4+ T cells, but it also involves other complex issues including adaptive responses within secondary lymphoid organs and within the graft itself. CD4+Foxp3+ regulatory T cells (Treg) are central to the maintenance of self-tolerance, the regulation of excessive immune responses, and experimental therapies use their immunoregulatory properties to suppress alloimmunity and to induce immunological tolerance towards the allograft. However, Treg based therapies are hindered by their instability and their potential to dedifferentiate into effector cells that can initiate/cause rejection. Our previous studies demonstrated that DEPTOR is expressed by CD4+ Treg cells, is markedly reduced upon mitogen activation, and that sustained expression of DEPTOR stabilizes lineage commitment via the epigenetic regulation of the Foxp3 promoter and by augmenting oxidative metabolism. In vivo, induced DEPTOR expression within Tregs is sufficient to promote immunoregulation and inhibit cardiac allograft rejection. This proposal tests the hypothesis that DEPTOR is a central modulator of Treg subset homeostasis and function, and that it is essential to maintain immunoregulation and tolerance following transplantation. It will also test whether DEPTOR functions via a novel regulatory signaling network and/or novel interactions with target genes within distinct Treg lineages or subsets. The approach is divided into three specific aims to: first, investigate mechanisms of DEPTOR function in Tregs and mTOR-independent targets of DEPTOR activity; second, systematically discover the extended DEPTOR-induced modulatory signaling network that enhances Treg function and lineage commitment; and third, identify small molecules that modulate DEPTOR protein expression, its downstream target genes and/or regulatory signaling activity as potential pro-tolerogenic therapeutic drugs. During the mentored phase of the K99, the results of the proposed studies will identify new roles for DEPTOR in Treg subsets and discover mTOR-independent effects of DEPTOR on Treg biology. These studies will launch the R00 phase of independent research to investigate the mechanistic function of the network of negative modulators and/or target genes in Treg subsets, and develop a pharmacological screening assay to identify small molecules that have potential as therapeutic drugs to induce immunological tolerance towards allografts and/or as a treatment for autoimmune disease. The broader implication of this work relates to the biological importance of Treg targeting as a component of tumor immunotherapy.